The present invention relates to novel peptide fragments [targeting signal] that are obtainable from the C-terminal region [C-terminal extension or C-terminal peptide fragment] of plant vacuole proteins and that, in operable linkage with any desired protein molecule, ensure that the proteins associated with those peptide fragments are directed or targeted specifically into the plant vacuole.
The present invention relates also to DNA sequences that code for the peptide fragments characterised in greater detail above and that, in operable linkage with any desired expressible DNA, result in a gene product that is directed specifically into the plant vacuole, and to mutants and variants thereof.
The present invention relates furthermore to recombinant DNA molecules that comprise the DNA sequence according to the invention in operable linkage with an expressible DNA, and to the vectors derived therefrom. Also included are host cells and/or host organisms, including transgenic plants, that comprise the said recombinant DNA or the vectors derived therefrom.
The present invention relates also to processes for the production of the DNA sequences according to the invention and of the recombinant DNA molecules and vectors comprising those DNA sequences, and to the use thereof for the production of transgenic plants.
In genetic engineering, there has recently been an increasing amount of interest in going beyond the pure expression of an inserted foreign gene to identify DNA sequences coding for so-called signal sequences that allow the associated gene product, according to its function, to reach its specific destination, where it can then develop its optimum activity or be stored in suitable manner. With regard to the plant as a whole, this means that attempts are being made, for example, to identify or develop promoters that permit tissue- and/or development-specific expression of an inserted foreign gene.
The target- or destination-oriented placing of inserted foreign genes or their expression products is not only of relevance at the level of the plant, however, but may also be of great importance even at cell level, especially as regards the effectiveness of the transformation.
For example, it is known that, in plant as well as in other eukaryotic cells, although the majority of proteins are synthesised on cytoplasmic ribosomes, a large number of those proteins are required in quite different subcellular compartments. The only exception is formed by some mitochondrial and chloroplast proteins, which are produced at the location in which they are used. The cytoplasmically produced proteins, on the other hand, are either transported along the endomembrane system passing through the cell into the lytic compartment [vacuole, lysosome] of the cell and into the extracellular space, or are taken up directly by their particular compartment [vacuole, chloroplast, peroxisome].
For the maintenance of this compartmentalisation at subcellular level, there must be specific transport and sorting systems inside the cell which ensure that the cytoplasmically produced proteins are distributed according to their function. These proteins must therefore contain one or more additional items of information which enable the said transport and sorting systems of the cell to recognise their own particular substrate and direct it to their specific destination. Thus, for example, in a great many cytoplasmic precursors of mitochondrial and chloroplast proteins it has been possible to detect at the N-terminal end a so-called transit peptide which ensures that the proteins are taken up into their particular compartment. Similarly, the nuclear proteins have a cell-nucleus-specific sequence.
Of particular importance for intracellular protein transport is the cell's endomembrane system. This membrane system, which passes through the cell and is composed of the endoplasmic reticulum and the Golgi apparatus, serves essentially to transport proteins, especially cytoplasmically produced proteins, to the lytic compartment [vacuole, lysosome] and to the extracellular space.
Proteins transported via the endomembrane system first pass into the endoplasmic reticulum. The necessary transport signal for this step is represented by a signal sequence at the N-terminal end of the molecule, the so-called signal peptide. As soon as this signal peptide has fulfilled its function, it is split off proteolytically from the precursor protein. By virtue of its specific function, this type of signal peptide sequence has been conserved to a high degree during evolution in all living cells, irrespective of whether they are bacteria, yeasts, fungi, animals or plants.
A further sorting step then takes place in the Golgi apparatus, where separation of the proteins intended for the lytic compartment [vacuole, lysosome] and of the proteins intended for secretion into the, extracellular space takes place. In experiments on yeasts and animals it has been found that proteins that do not contain an additional signal are apparently secreted automatically into the extraceilular space, while proteins that do contain such an additional sorting signal are discharged into the lytic compartment.
The nature of this sorting signal can vary very widely. In the animals studied hitherto, for example, it is generally a specific modification within the glycan chain of glycoproteins, namely a mannose 6-phosphate group. This group is recognised by a specific mannose 6-phosphate receptor, with the result that the corresponding proteins in specific vesicles are freed from the Golgi apparatus and transported to the lysosomes. However, it has hitherto not been possible to determine which polypeptide sequence gives the impetus for phosphorylation of a mannose group in the glycan side chain.
In yeasts, the corresponding sorting signal for the lytic compartment is not a glycan chain but an amino acid sequence which, after splitting off of the signal peptide, forms the N-terminus of the protein. This N-terminal targeting signal for the vacuole is generally split off in the vacuole itself by means of proteinase A. Often, the protein transported into the vacuole becomes a catalytically active enzyme only as a result of this splitting off of the targeting signal.
In plants, the question of the targeting signal responsible for directing proteins into the vacuole is of particular interest--especially from the point of view of application--because the vacuole not only represents the lytic compartment of the plant cell, but also forms the largest storage compartment for reserve substances, detoxification products and defence substances [Boler and Wiemken (1986)].
It would therefore be very advantageous to be able to direct proteins associated with an improvement in a plant's nutrient content specifically into the vacuole and store them there, since that is by far the largest compartment in the plant cell for dissolved substances. The most important storage proteins of tubers, bulbs, roots and stems, for example, are located in the vacuoles of the cells that compose those organs [Boiler and Wiemke (1986)]. Moreover, the storage proteins of most seeds are located in so-called protein bodies, specialised vacuoles to which the same targeting signals would seem to apply as to the vacuoles of the vegetative organs.
Similar considerations also apply to substances that can be used in the control of pests or diseases, especially when those substances prove to be toxic to the plant itself. It is therefore advantageous to deposit those substances in the plant vacuole too. Finally, in certain cases the vacuole also serves as a detoxification organ by, for example, storing the detoxification products synthesised by the plant [Boller and Wiemke (1986)]. Attempts are therefore being made to direct detoxification enzymes specifically into the vacuole too.
However, precisely the opposite problem arises, for example, in the case of attack of cultivated plants by certain fungal pathogens. These pathogens infect their host plant by spreading their mycelium through the plant's intercellular spaces. Since the chitinases and glucanases that can be used for controlling these pathogens are very often located in the vacuole, their bioavailability in the intercellular space is naturally very limited. In that case, therefore, it would be desirable to be able to discharge those proteins specifically into the extracellular compartment in order to increase the bioavailability of those substances in the intercellular space and thus permit effective control of the fungal pathogen.
Secretion into the extracellular space is also advantageous when specific substances are to be produced using cell cultures. In that case, the directed foreign proteins can very easily be isolated from the surrounding medium since they are secreted into the extracellular space. The breaking up of the cells that is necessary in the case of intracellular production can be omitted.
However, it has hitherto not been possible to identify, let alone isolate such a targeting signal for the plant vacuole.
On the assumption that an N-terminal targeting signal comparable to that in yeasts could be responsible in plants for directing proteins into the vacuole, Tague and Chrispeels (1987) linked various parts of the phytohaemagglutinin molecule from peas with a reporter gene and tested them in yeasts. It was found that in the yeast host, also in the case of phytohaemagglutinin, the N-terminal part of the molecule acts as a targeting signal for the vacuole.
A further vacuole protein, basic .beta.-1,3-glucanase, is synthesised with a C-terminal extension which is lost as the molecule matures. The same also applies to the lectins from rice, barley and wheat (wheatgerm agglutinin). The function of this extension was hitherto unknown.
Since a comparison of the C-termini of various proteins from the vacuole hitherto revealed no apparent homologies in this field, the hypothesis hitherto favoured was that, as with the yeasts, the decisive signal activity in plants originates in the N-terminus of those vacuole proteins.
One of the main problems to be solved within the scope of this invention was, therefore, to identify and isolate a peptide fragment [targeting sequence] responsible for directing an associated protein molecule specifically into the vacuole of the plant cell, and also the DNA sequence coding for the said peptide fragment.
Surprisingly, within the scope of the present invention it has now been possible to solve this problem by the use of procedures of which some are known.